Base-modified enzymatic nucleic acid

ABSTRACT

Method to produce a more active ribozyme by introducing a modified base into a substrate binding arm of the ribozyme or its catalytic core.

This application is a continuation of Usman, et al, “Base ModifiedEnzymatic Nucleic Acid”, U.S. Ser. No. 08/435,521, filed May. 5, 1995,now U.S. Pat. No. 5,767,263 which is a continuation-in-part ofMcSwiggen, “Optimization of Ribozyme Activity”, U.S. Ser. No.07/963,322, filed Oct. 15, 1992 and Usman et al., entitled“Base-modified enzymatic nucleic acid”, U.S. Ser. No. 08/149,210 (filedNov. 8, 1993), the whole of both of which, including drawings, is herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to enzymatic RNA molecules or ribozymes having amodified nucleotide base sequence.

Ribozymes are RNA molecules having an enzymatic activity which is ableto repeatedly cleave other separate RNA molecules in a nucleotide basesequence specific manner. Such enzymatic RNA molecules can be targetedto virtually any RNA transcript, and efficient cleavage achieved invitro. Kim et al., 84 Proc. Nati. Acad. Sci. USA 8788, 1987; Haseloffand Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; andJefferies et al., 17 Nucleic Acids Research 1371, 1989.

Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic portion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

By “complementarity” is meant a nucleic acid that can form hydrogenbond(s) with other RNA sequence by either traditional Watson-Crick orother non-traditional types (for example, Hoogsteen type) of base-pairedinteractions.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the effective concentration of ribozyme necessary to effect atherapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base pairing mechanism of binding, but also on the mechanismby which the molecule inhibits the expression of the RNA to which itbinds. That is, the inhibition is caused by cleavage of the RNA targetand so specificity is defined as the ratio of the rate of cleavage ofthe targeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base pairing. Thus, it is thought that the specificity ofaction of a ribozyme is greater than that of antisense oligonucleotidebinding the same RNA site.

The following discussion of relevant art is dependent on the diagramshown in FIG. 1, in which the numbering of various nucleotides in ahammerhead ribozyme is provided. This is not to be taken as anindication that the Figure is prior art to the pending claims, or thatthe art discussed is prior art to those claims.

Odai et al., FEBS 1990, 267:150, state that substitution of guanosine(G) at position 5 of a hammerhead ribozyme for inosine greatly reducescatalytic activity, suggesting “the importance of the 2-amino group ofthis guanosine for catalytic activity.”

Fu and McLaughlin, Proc. Natl. Acad. Sci. ( USA) 1992, 89:3985, statethat deletion of the 2-amino group of the guanosine at position 5 of ahammerhead ribozyme, or deletion of either of the 2′-hydroxyl groups atposition 5 or 8, resulted in ribozymes having a decrease in cleavageefficiency.

Fu and McLaughlin, Biochemistry 1992, 31:10941, state that substitutionof 7-deazaadenosine for adenosine residues in a hammerhead ribozyme cancause reduction in cleavage efficiency. They state that the “resultssuggest that the N⁷-nitrogen of the adenosine (A) at position 6 in thehammerhead ribozyme/substrate complex is critical for efficient cleavageactivity.” They go on to indicate that there are five criticalfunctional groups located within the tetrameric sequence GAUG in thehammerhead ribozyme.

Slim and Gait, 1992, BBRC 183, 605, state that the substitution ofguanosine at position 12, in the core of a hammerhead ribozyme, withinosine inactivates the ribozyme.

Tuschl et al., 1993 Biochemistry 32, 11658, state that substitution ofguanosine residues at positions 5, 8 and 12, in the catalytic core of ahammerhead, with inosine, 2-aminopurine, xanthosine, isoguanosine ordeoxyguanosine cause significant reduction in the catalytic efficiencyof a hammerhead ribozyme.

Fu et al., 1993 Biochemistry 32, 10629, state that deletion of guanineN⁷, guanine N² or the adenine N⁶-nitrogen within the core of ahammerhead ribozyme causes significant reduction in the catalyticefficiency of a hammerhead ribozyme.

Grasby et al., 1993 Nucleic Acids Res. 21, 4444, state that substitutionof guanosine at positions 5, 8 and 12 positions within the core of ahammerhead ribozyme with O⁶-methylguanosine results in an approximately75-fold reduction in k_(cat).

Seela et al., 1993 Helvetica Chimica Acta 76, 1809, state thatsubstitution of adenine at positions 13, 14 and 15, within the core of ahammerhead ribozyme, with 7-deazaadenosine does not significantlydecrease the catalytic efficiency of a hammerhead ribozyme.

Adams et al., 1994 Tetrahedron Letters 35, 765, state that substitutionof uracil at position 17 within the hammerhead ribozyme•substratecomplex with 4-thiouridine results in a reduction in the catalyticefficiency of the ribozyme by 50 percent.

Ng et al., 1994 Biochemistry 33, 12119, state that substitution ofadenine at positions 6, 9 and 13 within the catalytic core of ahammerhead ribozyme with isoguanosine, significantly decreases thecatalytic activity of the ribozyme.

Jennings et al., U.S. Pat. No. 5,298,612, indicate that nucleotideswithin a “minizyme” can be modified. They state-

“Nucleotides comprise a base, sugar and a monophosphate group.Accordingly, nucleotide derivatives or modifications may be made at thelevel of the base, sugar or monophosphate groupings . . . Bases may besubstituted with various groups, such as halogen, hydroxy, amine, alkyl,azido, nitro, phenyl and the like.”

SUMMARY OF THE INVENTION

This invention relates to production of enzymatic RNA molecules orribozymes having enhanced or reduced binding affinity and enhancedenzymatic activity for their target nucleic acid substrate by inclusionof one or more modified nucleotides in the substrate binding portion ofa ribozyme such as a hammerhead, hairpin, VS ribozyme or hepatitis deltavirus derived ribozyme. Applicant has recognized that only small changesin the extent of base-pairing or hydrogen bonding between the ribozymeand substrate can have significant effect on the enzymatic activity ofthe ribozyme on that substrate. Thus, applicant has recognized that asubtle alteration in the extent of hydrogen bonding along a substratebinding arm of a ribozyme can be used to improve the ribozyme activitycompared to an unaltered ribozyme containing no such altered nucleotide.Thus, for example, a guanosine base may be replaced with an inosine toproduce a weaker interaction between a ribozyme and its substrate, or auracil may be replaced with a bromouracil (BrU) to increase the hydrogenbonding interaction with an adenosine. Other examples of alterations ofthe four standard ribonucleotide bases are shown in FIGS. 6a-d withweaker or stronger hydrogen bonding abilities shown in each figure.

In addition, applicant has determined that base modification within somecatalytic core nucleotides maintains or enhances enzymatic activitycompared to an unmodified molecule. Such nucleotides are noted in FIG.7. Specifically, referring to FIG. 7, the preferred sequence of ahammerhead ribozyme in a 5′ to 3′ direction of the catalytic core is CUGANG A G•C GAA A, wherein N can be any base or may lack a base (abasic);G•C is a base-pair. The nature of the base-paired stem II (FIGS. 1, 2and 7) and the recognition arms of stems I and III are variable. In thisinvention, the use of base-modified nucleotides in those regions thatmaintain or enhance the catalytic activity and/or the nucleaseresistance of the hammerhead ribozyme are described. (Bases which can bemodified include those shown in capital letters).

Examples of base-substitutions useful in this invention are shown inFIGS. 6, 8-14. In preferred embodiments cytidine residues aresubstituted with 5-alkylcytidines (e.g., 5-methylcytidine, FIG. 8,R═CH₃, 9), and uridine residues with 5-alkyluridines (e.g.,ribothymidine (FIG. 8, R═CH₃, 4) or 5-halouridine (e.g., 5-bromouridine,FIG. 8, X═Br, 13) or 6-azapyrimidines (FIG. 8, 17) or 6-alkyluridine(FIG. 14). Guanosine or adenosine residues may be replaced bydiaminopurine residues (FIG. 8, 22) in either the core or stems. Inthose bases where none of the functional groups are important in thecomplexing of magnesium or other functions of a ribozyme, they areoptionally replaced with a purine ribonucleoside (FIG. 8, 23), whichsignificantly reduces the complexity of chemical synthesis of ribozymes,as no base-protecting group is required during chemical incorporation ofthe purine nucleus. Furthermore, as discussed above, base-modifiednucleotides may be used to enhance the specificity or strength ofbinding of the recognition arms with similar modifications.Base-modified nucleotides, in general, may also be used to enhance thenuclease resistance of the catalytic nucleic acids in which they areincorporated. These modifications within the hammerhead ribozyme motifare meant to be non-limiting example. Those skilled in the art willrecognize that other ribozyme motifs with similar modifications can bereadily synthesized and are within the scope of this invention.

Substitutions of sugar moieties as described in the art cited above, mayalso be made to enhance catalytic activity and/or nuclease stability.

Thus, in a first aspect, the invention features a modified ribozymehaving one or more substrate binding arms including one or more modifiednucleotide bases; and in a related aspect, the invention features amethod for production of a more active modified ribozyme (compared to anunmodified ribozyme) by inclusion of one or more of such modifiednucleotide bases in a substrate binding arm.

The invention provides ribozymes having increased enzymatic activity invitro and in vivo as can be measured by standard kinetic assays. Thus,the kinetic features of the ribozyme are enhanced by selection ofappropriate modified bases in the substrate binding arms. Applicantrecognizes that while strong binding to a substrate by a ribozymeenhances specificity, it may also prevent separation of the ribozymefrom the cleaved substrate. Thus, applicant provides means by whichoptimization of the base pairing can be achieved. Specifically, theinvention features ribozymes with modified bases with enzymatic activityat least 1.5 fold (preferably 2 or 3 fold) or greater than theunmodified corresponding ribozyme. The invention also features a methodfor optimizing the kinetic activity of a ribozyme by introduction ofmodified bases into a ribozyme and screening for those with higherenzymatic activity. Such selection may be in vitro or in vivo. Byenchanced activity is meant to include activity measured in vivo wherethe activity is a reflection of both catalytic activity and ribozymestability. In this invention, the product of these properties inincreased or not significantly (less that 10 fold) decreased in vivocompared to an all RNA ribozyme.

By “enzymatic portion” is meant that part of the ribozyme essential forcleavage of an RNA substrate.

By “substrate binding arm” is meant that portion of a ribozyme which iscomplementary to (i.e., able to base-pair with) a portion of itssubstrate. Generally, such complementarity is 100%, but can be less ifdesired. For example, as few as 10 bases out of 14 may be base-paired.Such arms are shown generally in FIGS. 1-3 as discussed below. That is,these arms contain sequences within a ribozyme which are intended tobring ribozyme and target RNA together through complementarybase-pairing interactions; e.g., ribozyme sequences within stems I andIII of a standard hammerhead ribozyme make up the substrate-bindingdomain (see FIG. 1).

By “unmodified nucleotide base” is meant one of the bases adenine,cytosine, guanosine, uracil joined to the 1′ carbon ofβ-D-ribo-furanose. The sugar also has a phosphate bound to the 5′carbon. These nucleotides are bound by a phosphodiester between the 3′carbon of one nucleotide and the 5′ carbon of the next nucleotide toform RNA.

By “modified nucleotide base” is meant any nucleotide base whichcontains a modification in the chemical structure of an unmodifiednucleotide base which has an effect on the ability of that base tohydrogen bond with its normal complementary base, either by increasingthe strength of the hydrogen bonding or by decreasing it (e.g., asexemplified above for inosine and bromouracil). Other examples ofmodified bases include those shown in FIGS. 6a-d and other modificationswell known in the art, including heterocyclic derivatives and the like.

In preferred embodiments the modified ribozyme is a hammerhead, hairpinVS ribozyme or hepatitis delta virus derived ribozyme, and thehammerhead ribozyme includes between 32 and 40 nucleotide bases. Theselection of modified bases is most preferably chosen to enhance theenzymatic activity (as observed in standard kinetic assays designed tomeasure the kinetics of cleavage) of the selected ribozyme, i.e., toenhance the rate or extent of cleavage of a substrate by the ribozyme,compared to a ribozyme having an identical nucleotide base sequencewithout any modified base.

By “enzymatic nucleic acid molecule” it is meant a nucleic acid moleculewhich has complementarity in a substrate binding region to a specifiedgene target, and also has an enzymatic activity which is active tospecifically cleave RNA in that target. That is, the enzymatic nnucleicacid molecule is able to inter-molecularly cleave RNA and therebyinactivate a target RNA molecule. This complementarity functions toallow sufficient hybridization of the enzymatic nucleic acid molecule tothe target RNA to allow the cleavage to occur. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% mayalso be useful in this invention.

In preferred embodiments of this invention, the enzymatic nucleic acidmolecule is formed in a hammerhead or hairpin motif, but may also beformed in the motif of a hepatitis delta virus, group I intron or RNasePRNA (in association with an RNA guide sequence) or Neurospora VS RNA.Examples of such hammerhead motifs are described by Rossi et al., 1992,Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampelet al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, andHampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of thehepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983Cell 35, 849, Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799) and of the Group I intron by Cech et al.,U.S. Pat. No. 4,987,071. These specific motifs are not limiting in theinvention and those skilled in the art will recognize that all that isimportant in an enzymatic nucleic acid molecule of this invention isthat it has a specific substrate binding site which is complementary toone or more of the target gene RNA regions, and that it have nucleotidesequences within or surrounding that substrate binding site which impartan RNA cleaving activity to the molecule.

In a preferred embodiment the invention provides a method for producinga class of enzymatic cleaving agents which exhibit a high degree ofspecificity for the RNA of a desired target. The enzymatic nucleic acidmolecule is preferably targeted to a highly conserved sequence region ofa target RNAs such that specific treatment of a disease or condition canbe provided with either one or several enzymatic nucleic acids. Suchenzymatic nucleic acid molecules can be delivered exogenously tospecific cells as required.

By “kinetic assays” or “kinetics of cleavage” is meant an experiment inwhich the rate of cleavage of target RNA is determined. Often a seriesof assays are performed in which the concentrations of either ribozymeor substrate are varied from one assay to the next in order to determinethe influence of that parameter on the rate of cleavage.

By “rate of cleavage” is meant a measure of the amount of target RNAcleaved as a function of time.

In a second aspect, enzymatic nucleic acid having a hammerheadconfiguration and modified bases which maintain or enhance enzymaticactivity is provided. Such nucleic acid is also generally more resistantto nucleases than unmodified nucleic acid. By “modified bases” in thisaspect is meant those shown in FIGS. 6 A-D, and 8, or their equivalents;such bases may be used within the catalytic core of the enzyme as wellas in the substrate-binding regions. As noted above, substitution in thecore may decrease in vitro activity but enhances stability. Thus, invivo the activity may not be significantly lowered. As exemplifiedherein such ribozymes are useful in vivo even if active over all isreduced 10 fold. Such ribozymes herein are said to “maintain” theenzymatic activity on all RNA ribozyme.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be ≧2 base-pair long. Each N isindependently any base or non-nucleotide as used herein. (SEQ ID NO:1-2)

FIGS. 2, (a-d) (a) is a diagrammatic representation of the hammerheadribozyme domain known in the art; FIG. (b)is a diagrammaticrepresentation of the hammerhead ribozyme as divided by Uhlenbeck (1987,Nature, 327, 596-600) into a substrate and enzyme portion; FIG. (c) is asimilar diagram showing the hammerhead divided by Haseloff and Gerlach(1988, Nature, 334, 585-591) into two portions; and FIG. (d) is asimilar diagram showing the hammerhead divided by Jeffries and Symons(1989, Nucl. Acids. Res., 17, 1371—1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of ahairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs(i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided oflength 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 ormore). Helix 2 and helix 5 may be covalently linked by one or more bases(i.e., r is ≧1 base). Helix 1, 4 or 5 may also be extended by 2 or morebase pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure,and preferably is a protein binding site. In each instance, each N andN′ independently is any normal or modified base and each dash representsa potential base-pairing interaction. These nucleotides may be modifiedat the sugar, base or phosphate. Complete base-pairing is not requiredin the helices, but is preferred. Helix 1 and 4 can be of any size(i.e., o and p is each independently from 0 to any number, e.g., 20) aslong as some base-pairing is maintained. Essential bases are shown asspecific bases in the structure, but those in the art will recognizethat one or more may be modified chemically (abasic, base, sugar and/orphosphate modifications) or replaced with another base withoutsignificant effect. Helix 4 can be formed from two separate molecules,i.e., without a connecting loop. The connecting loop when present may bea ribonucleotide with or without modifications to its base, sugar orphosphate. “q” is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H, refers to bases A, U or C. Yrefers to pyrimidine bases.” (SEQ ID NO: 3-4)

FIG. 4 is a representation of the general structure of the hepatitisdelta virus ribozyme domain known in the art. (SEQ ID NO: 5)

FIG. 5 is a representation of the general structure of the self-cleavingVS RNA ribozyme domain. (SEQ ID NO: 6)

FIGS. 6a-d are diagrammatic representations of standard basemodifications for (a) adenine,(b) guanine,(c) cytosine and (d) uracil,respectively.

FIG. 7 is a diagrammatic representation of a position numberedhammerhead ribozyme (according to Hertel et al., Nucleic Acids Res.1992, 20:3252) showing specific substitutions in the catalytic core andsubstrate binding arms. (SEQ ID NO: 1-2) Compounds 4, 9, 13, 17, 22, 23are described in FIG. 8.

FIG. 8 is a diagrammatic representation of various nucleotides that canbe substituted in the catalytic core of a hammerhead ribozyme.

FIG. 9 is a diagrammatic representation of the synthesis of aribothymidine phosphoramidite.

FIG. 10 is a diagrammatic representation of the synthesis of a5-methylcytidine phosphoramidite.

FIG. 11 is a diagrammatic representation of the synthesis of5-bromouridine phosphoramidite.

FIG. 12 is a diagrammatic representation of the synthesis of6-azauridine phosphoramidite.,

FIG. 13 is a diagrammatic representation of the synthesis of2,6-diaminopurine phosphoramidite.

FIG. 14 is a diagrammatic representation of the synthesis of a6-methyluridine phosphoramidite.

FIG. 15 is a representation of a hammerhead ribozyme targeted to site A(HHA). Site of 6-methyl U substitution is indicated. (SEQ ID NO: 7-8)

FIG. 16 shows RNA cleavage reaction catalyzed by HHA ribozyme containing6-methyl U-substitution (6-methyl-U4). U4, represents a HHA ribozymecontaining no 6-methyl-U substitution.

FIG. 17 is a representation of a hammerhead ribozyme targeted to site B(HHB). Sites of 6-methyl U substitution are indicated. (SEQ ID NO: 9-10)

FIG. 18 shows RNA cleavage reaction catalyzed by HHB ribozyme containing6-methyl U-substitutions at U4 and U7 positions (6-methyl-U4). U4,represents a HHB ribozyme containing no 6-methyl-U substitution.

FIG. 19 is a representation of a hammerhead ribozyme targeted to site C(HHC). Sites of 6-methyl U substitution are indicated. (SEQ ID NO:11-12)

FIG. 20 shows RNA cleavage reaction catalyzed by HHC ribozyme containing6-methyl U-substitutions at U4 and U7 positions (6-methyl-U4). U4,represents a HHC ribozyme containing no 6-methyl-U substitution.

FIG. 21 shows 6-methyl-U-substituted HHA ribozyme-mediated inhibition ofrat smooth muscle cell proliferation.

FIG. 22 shows 6-methyl-U-substituted HHC ribozyme-mediated inhibition ofstromelysin protein production in human synovial fibroblast cells.

Modified Ribozymes

There is a narrow range of binding free-energies between a ribozyme andits substrate that will produce maximal ribozyme activity. Such bindingenergy can be optimized by making ribozymes with G to I and U to BrUsubstitutions (or equivalent substitutions) in the substrate-bindingarms. This allows manipulation of the binding free-energy withoutactually changing the target recognition sequence, the length of the twosubstrate-binding arms, or the enzymatic portion of the ribozyme. Theshape of the free-energy vs. ribozyme activity curve can be readilydetermined using data from experiments in which each base (or severalbases) is modified or unmodified, and without the complication ofchanging the size of the ribozyme/substrate interaction.

Such experiments will indicate the most active ribozyme structure. It islikely that only one or two modifications are necessary since a verysmall change in binding free energy (even one base-pair interaction) candramatically affect ribozyme activity; the use of modified bases thuspermits “fine tuning” of the binding free energy to assure maximalribozyme activity. In addition, replacement of such bases, e.g., I forG, may permit a higher level of substrate specificity when cleavage ofnon-target RNA is a problem.

Method

Modified substrate binding arms can be: synthesized using standardmethodology. For example, phosphoramidites of inosine and 5-bromouracilcan be used. Generally, a target site that has been optimized for stem Iand III lengths (in a hammerhead ribozyme—other ribozymes can be treatedin a similar manner), and that has G and/or U in the ribozyme portion ofstem I and III, is selected. Modified ribozymes are made by replacingvarious G and U residues with I and BrU, respectively, during synthesisof the ribozyme. The modified ribozymes are then tested to determinekinetic parameters using standard procedures (see McSwiggen, “ImprovedRibozymes”, U.S. Ser. No. 07/884,521, filed May 14, 1992, herebyincorporated by reference herein). The binding affinities for theribozymes can also be determined by standard procedures, e.g., byT-melt, gel-binding, or by competition kinetics assays. By comparison ofbinding affinity and ribozyme activity the optimum binding affinity of aribozyme can then be found. Other combinations of G, I, U BrU, and otherbases can then be tested with nearly identical binding free energy, butdifferent base sequence, to determine whether factors other than simplebinding free-energy play a role.

It is preferred to perform routine experiments of this type to select adesired ribozyme substrate binding sequence by use of an unmodifiedribozyme with a modified substrate (which contains the modified bases).That is, the reverse experiment to that described above is performed.Such an experiment is more readily performed since the substrate isgenerally shorter than the ribozyme, and can be readily synthesizedwithout concern about its secondary structure. Thus, a single ribozymecan be tested against a plurality of modified substrates in order todefine which of the substrates provides better kinetic results. Once apreferred substrate is identified, the ribozyme can then be modified ina way which mirrors the selected substrate, and then tested against anunmodified substrate.

Such experiments will define useful ribozymes of this invention in whichone or more modified bases are provided in the substrate binding armswith greater enzymatic activity in vitro and in vivo than comparableunmodified ribozymes. Such modifications may also be advantageous ifthey increase the resistance of a ribozyme to enzymatic degradation invivo.

Synthesis of Ribozymes

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzymatic nucleicacid motifs (e.g., of the hammerhead or the hairpin structure) are usedfor exogenous delivery. The simple structure of these moleculesincreases the ability of the enzymatic nucleic acid to invade targetedregions of the mRNA structure.

The ribozymes are chemically synthesized. The method of synthesis usedfollows the procedure for normal RNA synthesis as described in Usman etal., 1987 J. Am. Chem. Soc., 109, 7845 and in Scaringe et al., 1990Nucleic Acids Res., 18, 5433 and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. The average stepwise coupling yieldswere >98%.

Ribozymes are purified by gel electrophoresis using general methods orare purified by high pressure liquid chromatography (HPLC; See Usman etal., Synthesis, deprotection, analysis and purification of RNA andribozymes, filed May, 18, 1994, U.S. Ser. No. 08/245,736 the totality ofwhich is hereby incorporated herein by reference) and are resuspended inwater.

Various modifications to ribozyme structure can be made to enhance theutility of ribozymes. Such modifications will enhance shelf-life,half-life in vitro, stability, and ease of introduction of suchribozymes to the target site, e.g., to enhance penetration of cellularmembranes, and confer the ability to recognize and bind to targetedcells.

Optimizing Ribozyme Activity

Ribozyme activity can be optimized as described by Stinchcomb et al.,“Method and Composition for Treatment of Restenosis and Cancer UsingRibozymes,” filed May 18, 1994, U.S. Ser. No. 08/245,466. The detailswill not be repeated here, but include altering the length of theribozyme binding arms (stems I and III, see FIG. 2c), or chemicallysynthesizing ribozymes with modifications that prevent their degradationby serum ribonucleases (see e.g., Eckstein et al., InternationalPublication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565;Pieken et al., 1991 Science 253, 314; Usman and Cedergren, 1992 Trendsin Biochem. Sci. 17, 334; Usman et al., International Publication No. WO93/15187; and Rossi et al., International Publication No. WO 91/03162,as well as Usman, N. et al. U.S. patent application Ser. 07/829,729, andSproat, European Patent Application 92110298.4 which describe variouschemical modifications that can be made to the sugar moieties ofenzymatic RNA molecules. Modifications which enhance their efficacy incells, and removal of stem II bases to shorten RNA synthesis times andreduce chemical requirements. (All these publications are herebyincorporated by reference herein.).

Administration of Ribozyme

Sullivan et al., PCT WO94/02595, describes the general methods fordelivery of enzymatic RNA molecules . Ribozymes may be administered tocells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. For some indications, ribozymes may be directly deliveredex vivo to cells or tissues with or without the aforementioned vehicles.Alternatively, the RNA/vehicle combination is locally delivered bydirect injection or by use of a catheter, infusion pump or stent. Otherroutes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan et al., supra and Draper etal., PCT WO 93/23569 which have been incorporated by reference herein.

EXAMPLES

The following are non-limiting examples showing the synthesis ofbase-modified catalytic nucleic acids.

Example 1

Synthesis of Hammerhead Ribozymes Containing Base-Modified Nucleotides

The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al., J. Am. Chem. Soc. 1987, 109,7845-7854 and in Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441and makes use of common nucleic acid protecting and coupling groups,such as dimethoxytrityl at the 5′-end, and phosphoramidites at the3′-end (compounds 4, 9, 13, 17, 22, 23). The average stepwise couplingyields were >98%. These base-modified nucleotides may be incorporatednot only into hammerhead ribozymes, but also into hairpin, VS ribozymes,hepatitis delta virus, or Group 1 or Group 2 introns. They are,therefore, of general use as replacement motifs in any nucleic acidstructure.

In the case of the hammerhead ribozyme the following specificsubstitutions may be used:

Referring to FIG. 7, in the catalytic core (numbered nucleotides), thepyrimidine C3 may be replaced by the cytosine analogs shown in FIG. 6cand compound 9 in FIG. 8.

Referring to FIG. 7, in the catalytic core (numbered nucleotides), thepyrimidines U4 and N7 may be replaced by the cytosine analogs shown inFIG. 4c, the uridine analogs shown in FIG. 6d and compounds 4, 9, 13 and17 in FIG. 8 and FIG. 14.

Referring to FIG. 7, in the catalytic core (numbered nucleotides), thepurines G5, G8 and G12 may be replaced by the guanine analogs shown inFIG. 6b and compounds 22 and 23 in FIG. 8.

Referring to FIG. 7, in the catalytic core (numbered nucleotides), thepurines A6, A9, A13 and A14 may be replaced by the adenine analogs shownin FIG. 6a and compounds 22 and 23 in FIG. 8.

Referring to FIGS. 1 and 5, in stems I, II and III any of thepyrimidines may be replaced by the pyrimidine analogs shown in FIGS. 6cand 6 dand compounds 4, 9, 13 and 17 in FIG. 8 and FIG. 14 as long asbase-pairing is maintained in the stems.

Referring to FIGS. 12 and 7, in stems I, II and III any of the purinesmay be replaced by the purine analogs shown in FIGS. 6a and 6 bandcompounds 22 and 23 in FIG. 8 as long as base-pairing is maintained inthe stems.

Referring to FIGS. 12 and 7, in loop II (denoted as loop II in FIG. 7)any nucleotide may be replaced by the pyrimidine analogs shown in FIGS.6c and 6 d, the purine analogs shown in FIGS. 6a and 6 b and compounds4, 9, 13, 17, 22 and 23 in FIG. 8 and FIG. 14.

Example 2

Synthesis of Ribothymidine Phosphoramidite 4

Referring to FIG. 9, ribothymidine 1 was prepared according toVorbrüggen et al., Chem. Ber. 1981, 114:1234, and tritylated to yieldDMT derivative 2. 2 was silylated to yield 2′-O-TBDMS derivative 3. Thephosphoramidite 4 was prepared according to Scaringe et al., NucleicAcids Res. 1990, 18:5433.

Example 3

Synthesis of 5-Methylcytidine Phosphoramidite 9

Referring to FIG. 10, Ribothymidine 1 (4 g, 15.5 mmol) was coevaporatedwith dry pyridine (2 x 100 ml) and redissolved in dry pyridine (100 ml).To the resulting solution 4,4′-DMT-Cl (6.3 g, 18.6 mmol) was added andthe reaction mixture was left at room temperature (about 20-25° C. for16 hours. The reaction mixture was quenched with methanol (25 ml) andevaporated to dryness. The residue was partitioned between chloroformand 5% sodium bicarbonate. The organic layer was washed with 5% sodiumbicarbonate and brine, then dried over sodium sulfate and evaporated.The residue was additionally dried by coevaporation with dry pyridine(2×50 ml) then redissolved in dry pyridine (100 ml) and acetic anhydride(4.4 ml, 46.5 mmol) was added to the resulting solution. The reactionmixture was left at room temperature overnight, then quenched withmethanol (25 ml), evaporated and worked-up as outlined above. The crude5′-O-dimethoxytrityl-2′,3′,-di-O-acetyl-ribo-thymidine 5 was purified byflash chromatography on silica gel,(hexanes:ethylacetate:triethylamine/45:45:10 to give 6.86 g (68.7%) of 5as a yellowish foam.

Triethylamine (14.72 ml, 105.6 mmol) was added dropwise to a stirredice-cooled mixture of triazole (6.56 g, 95.04 mmol) and phosphorousoxychloride (2 ml, 21.2 mmol) in 100 ml of dry acetonitrile. A solutionof nucleoside 5 (6.89, 10.56 mmol) in 50 ml of dry acetonitrile wasadded dropwise to the resulting suspension and the reaction mixture wasstirred at room temperature for 4 hours. The reaction was concentrated,dissolved in chloroform and washed with a saturated aqueous solution ofsodium bicarbonate, water, dried over sodium sulfate and evaporated todryness. To a solution of the residue (7.24 g) in dioxane (120 ml) wasadded 40 ml of 29% aqueous NH₄OH and the resulting solution was leftovernight, then evaporated to dryness to yield 6.86 g of crude cytidinederivative 6 which was used without purification.

To a solution of 6 (3.5 g, 6.25 mmol) in dry pyridine (100 ml) was added3.97 ml of trimethylchlorosilane to transiently protect free sugarhydroxyls. The reaction mixture was then treated with isobutyrylchloride (0.98 ml, 9.375 mmol) for 5 hours. The resulting mixture wasquenched with 10 ml of methanol, then 10 ml of water was added and after10 minutes 10 ml of 29% aq. ammonia was added and the reaction mixturewas stirred for 2 hours and evaporated to dryness. The resulting residuewas worked-up as outlined above for the compound 5 and purified by flashchromatography on silica gel (ethylacetate:hexanes/1:3) to yield 2.37 g(60%) of the nucleoside 7.

To a solution of compound 7 (1.3 g, 2.06 mmol) in dry pyridine 0.97 g(5.72 mmol) of silver nitrate-was added followed by 2.86 ml of a 1 Msolution of tert-butyidimethyl chloride in THF. The reaction mixture wasleft for 8 hours, evaporated, and dissolved in chloroform. The silversalt precipitate was filtered off and the reaction solution was washedwith 5% aq. sodium bicarbonate and brine, dried over sodium sulfate andevaporated. The mixture of 2′- and 3′-isomers was separated by flashchromatography on silica gel (hexanes:ethylacetate/4:1) to yield 0.62 g(40%) of 2′-isomer 8, which was converted to the phosphoramidite 9 bythe general method described in Scaringe et al., Nucleic Acids Res.1990, 18:5433.

Example 4

Synthesis of 5-Bromouridine Phosphoramidite 13 (See, Talbat et al.,Nucl. Acids Res. 18:3521-21, 1990)

Referring to FIG. 11, 5-Bromouridine 10 (1.615 g, 5 mmol) wascoevaporated with dry pyridine and redissolved in dry pyridine. To theresulting solution 2.03 g (6 mmol) of DMT-Cl was added and the reactionmixture was left overnight. After work-up and purification by flashchromatography on silica gel (chloroform:methanol/95:5) 2.5 g (80%) ofthe dimethoxytritylated compound 11 was obtained.

To a solution of 11 (2 g) in dry pyridine was added 1.5 eq. of TBDMS-Clfor 2 days. The reaction mixture was evaporated, dissolved inchloroform, washed with 5% aq. sodium bicarbonate and brine. The organiclayer was dried over sodium sulfate, evaporated and purified by flashchromatography on silica gel (ethylacetate:hexanes/1:2) to yield 1.4 g(60%) of 2′-isomer 12, which was converted to the phosphoramidite 13 bythe general method described in Scaringe et al., Nucleic Acids Res.1990, 18:5433.

Example 5

Synthesis of 6-azauridine Phosphoramidite 17

Referring to FIG. 12, 6-Azauridine (4.9 g, 20 mmol) was evaporated withdry pyridine (2×100 ml) and dissolved in dry pyridine (100 ml) and,after addition of 4,4′-DMT-Cl (7.45 g, 22 mmol) left for 16 hours atroom temperature. The reaction mixture was diluted with dry MeOH (50ml), evaporated to dryness, coevaporated with toluene (2×100 ml), theresidue dissolved in CHCl₃ (500 ml) and washed with 5% NaHCO₃ (100 ml),brine (100 ml), dried, and purified by flash chromatography (a gradientCHCl₃ to 5% EtOH/CHCl₃ to yield 1 g (92.2%) of intermediate) 15.

To a solution of 15 (3.23 g, 5.9 mmol) in 100 ml of dry THF, AgNO₃ (7.08mmol) and dry pyridine (2.1 ml, 4.4 mmol) were added. The reactionmixture was stirred at room temperature until full dissolution of AgNO₃(about 1 hour) occurred. Then 7 ml of a 1 M solution of TBDMS-Cl in THFwas added and the reaction mixture stirred for 16 hours at roomtemperature. The reaction mixture was filtered and the filtrateevaporated to dryness. The resulting residue was dissolved in CHCl₃ (300ml) and washed with 5% NaHCO₃ (100 ml), brine (100 ml), dried, andpurified by flash chromatography (gradient of hexanes to hexanes:ethylacetate/1:1) to yield 3.71 g (62%) of 2′-TBDMS-isomer 16 which wasconverted to the phosphoramidite 17 by the general method described inScaringe et al., Nucleic Acids Res. 1990, 18:5433.

Example 6

Synthesis of 2,6-diaminopurine Phosphoramidite 22

Referring to FIG. 13, phosphoramidite 22 was prepared by the generalmethod described in Scaringe et al., Nucleic Acids Res. 1990, 18:5433.Specifically, guanosine (11.32 g, 40 mmol) was dried by coevaporationwith dry pyridine and redissolved in dry pyridine. Chlorotrimethylsilane(26.4 ml, 208 mmol) was added under stirring to the above solution andthe reaction mixture was stirred overnight. To the resultingpersilylated guanosine derivative phenylacetylchloride (12.7 ml, 96mmol) was added dropwise and the reaction mixture was stirred for 12hours. The reaction was quenched with 50 ml of methanol and 50 ml ofwater and stirred for 15 minutes, then 50 ml of 29% ammonia was addedand the reaction mixture left for an additional 2 hours. Solvents wereremoved in vacuo, and the resulting oil was partitioned between ethylacetate and water. The separated water layer was washed with ethylacetate and was precipitated at 4° C. The resulting solid was filteredoff to give 8 g of N²phenylacetylguanosine 18. The mother liquor wasconcentrated to give additional crop (4 g). Overall yield˜12 g (75%).

N²Phenylacetylguanosine 18 (2.3 g, 5.73 mmol) was dried by coevaporation(3 times) with dry pyridine and dissolved in 50 ml of dry pyridine. Tothe resulting solution dimethoxytritylohloride (2.33 g, 6.88 mmol) wasadded and the reaction mixture was left at room temperature for 5 hours.The reaction was quenched with 25 ml of methanol and evaporated todryness. The residue was dissolved in dichloromethane, washed with 5%aq. sodium bicarbonate and brine, dried over sodium sulfate andevaporated. The resulting oil was further dried by coevaporation withdry pyridine, dissolved in pyridine and treated with acetic anhydride(1.4 ml) for 4 hours at room temperature. The reaction mixture wasquenched and worked-up as described above. The crude final compound waspurified by flash chromatography on silica gel usingdichloromethane:methanol/98:2 mixture as eluent. The desired fractionswere collected and evaporated to give 3.5 g (77%) of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N²-phenylacetylguanosin e 19 as ayellowish foam.

To a solution of compound 19 (3.5 g, 4.45 mmol) in 50 ml of drydichloromethane, containing 3.11 ml of dilsopropylethylamine, was addedmesitylenesulfonyl chloride (1.9 g, 8.9 mmol) and dimethylaminopyridine(0.28 g). The reaction mixture was stirred for 30 minutes, evaporatedand purified by flash chromatography on silica gel using dichloromethane(11) followed by 2% Methanol in dichloromethane (0.71) to give 2.8 g(64%) of O⁶-mesitylene intermediate 20. To a solution of 20 in 40 ml ofdry tetrahydrofuran lithium disulfide (0.3 g, 6.8 mmol) was added andthe reaction mixture was stirred for 20 hours. The resulting clearsolution was evaporated and worked-up as described above. The residuewas purified by flash chromatography on silica gel in 1% Methanol indichloromethane to give 1.1 g (31%) of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N²phenylacetyl-6-thiogua nosine21.

To an ice-cooled (0° C.) solution of5′-O-dimethoxytrityl-2′,3′-di-O-acetyl-N²phenylacetyl-6-thiogua nosine21 (1 g) in pyridine:methanol/20 ml:2.6 ml, 2.4 ml of 1M aq. sodiumhydroxide were added and the reaction mixture was allowed to stay at 0°C. for 20 minutes. The solution was neutralized with Dowex 2×8 (Pyr⁺) topH 7. The resin was filtered off and washed with aq. pyridine. Thecombined filtrate and washings were evaporated and dried in vacuo togive quantitatively 5′-O-dimethoxytrityl-N²phenylacetyl-6-thioguanosine.

To a stirred suspension of5′-O-dimethoxytrityl-N²phenylacetyl-6-thioguanosine (1.13 g, 1.57 mmol)in dry acetonitrile (35 ml) and triethylamine (1 ml) was addeddinitrofluorobenzene (0.34 g, 1.88 mmol) and the reaction mixture wasstirred under anhydrous conditions for 2 hours. The reaction wasevaporated and worked-up as described for compound 20 and purified byflash chromatography on silica gel in 1% methanol in chloroform(containing 1% triethylamine) as an eluent to give 0.93 g (67%) of5′-O-dimethoxytrityl-N²phenylacetyl-6-S-dinitrophenyl guanosine.

To a solution of 5′-O-dimethoxytrityl-N²phenylacetyl-6-S-dinitrophenylguanosine (0.9 g ,1 mmol) in dry pyridine t-butyldimethylsilylchloride(0.46 g, 3 mmol) and tetrabutylammonium nitrate (3 mmol) were added andthe reaction mixture was left for 50 hours. TLC (hexane:ethylacetate/3:1) showed disappearance of the starting material and formationof two new compounds with a predominance of a lower R_(f) (3′-O-silylisomer according to ¹H-NMR). The desired 2′-isomer (70 mg) was obtainedafter evaporation and work-up and separation by flash chromatography onsilica gel using hexane:ethyl acetate/4:1 as eluent. The remainingmixture was rearranged in methanol with 2 drops of triethylamine andseparated as above. This rearrangement procedure was repeated twice tofinally give 250 mg of the desired 2′-isomer.5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyl-N²phenylacetyl-6-S-dinitrophenylguanosine.

5′-O-Dimethoxytrityl-2′-O-t-butyidimethylsilyl-N²phenylacetyl-6-S-dinitrophenyl guanosine (0.18 g, 0.18 mmol) was dissolved in drytetrahydrofuran under dry argon. N-Methylimidazole (0.01 ml, 0.09 mmol)and sym-collidine (0.178 ml, 1.35 mmol) were added and the solution wasice-cooled. 2-Cyanoethyl N,N′-diisopropylchlorophosphoramidite (0.083ml, 0.36 mmol) was added dropwise and stirring was continued for 3 hoursat room temperature. The reaction mixture was again ice-cooled andquenched with 6 ml of dry degassed ethyl acetate. After 5 min stirringthe mixture was concentrated in vacuo (40° C.), dissolved in chloroform,washed with 5% aq sodium bicarbonate, then with brine and evaporated.The residue was purified by flash chromatography on silica gel usingethyl acetate:hexane/:3 containing 2% triethylamine as an eluent toyield 0.14 g (64%) 5′-O-dimethoxytrityl-2′-O-t-butyidimethylsilyl-N²phenylacetyl-6-S-dinitrophenylguanosine-3′-(2-cyanoethylN,N-diisopropylphosphoramidite) 22 as a yellow foam.

Example 7

Synthesis of 6-methyl-uridine phosphoramidite

Referring to FIG. 14, the suspension of 6-methyl-uracil (2.77 g, 21.96mmol) in the mixture of hexamethyidisilazane (50 mL) and dry pyridine(50 mL) was refluxed for three hours. The resulting clear solution oftrimethylsilyl derivative of 6-methyl uracyl was evaporated to drynessand coevaporated 2 times with dry toluene to remove traces of pyridine.To the solution of the resulting clear oil, in dry acetonitrile,1-O-acetyl-2′,3′,5′-tri-O-benzoyl-b-D-ribose (10.1 g, 20 mmol) was addedand the reaction mixture was cooled to 0° C. To the above stirredsolution, trimethylsilyl trifluoromethanesulfonate (4.35 mL, 24 mmol)was added dropwise and the reaction mixture was stirred for 1.5 h at 0°C. and then 1h at room temperature. After that the reaction mixture wasdiluted with dichloromethane washed with saturated sodium bicarbonateand brine. The organic layer was evaporated and the residue was purifiedby flash chromatography on silica gel with ethylacetate-hexane (2:1)mixture as an eluent to give 9.5 g (83%) of the compound 2 and 0.8 g ofthe corresponding N¹,N³-bis-derivative.

To the cooled (-10° C.) solution of the compound (4.2 g, 7.36 mmol) inthe mixture of pyridine (60 mL) and methanol (10 mL) ice-cooled 2Maqueous solution of sodium hydroxide (16 mL) was added with constantstirring. The reaction mixture was stirred at −10° C. for additional 30minutes and then neutralized to pH 7 with Dowex 50 (Py⁺). The resin wasfiltered off and washed with a 200 mL mixture of H₂O—Pyridine (4:1). Thecombined “mother liquor” and the washings were evaporated to dryness anddried by multiple coevaporation with dry pyridine. The residue wasredissolved in dry pyridine and then mixed with dimethoxytrityl chloride(2.99 g, 8.03 mmol). The reaction mixture was left overnight at roomtemperature. Reaction was quenched with methanol (25 mL) and the mixturewas evaporated. The residue was dissolved in dichloromethane, washedwith saturated aqueous sodium bicarbonate and brine. The organic layerwas dried over sodium sulfate and evaporated. The residue was purifiedby flash chromatography on silica gel using linear gradient of MeOH (2%to 5%) in CH₂Cl₂ as eluent to give 3.4 g (83%) of the compound 6.

Example 8

Synthesis of 6-methyl-cytidine phosphoramidite

Triethylamine (13.4 ml, 100 mmol) was added dropwise to a stirredice-cooled mixture of 1,2,4-triazole (6.22g, 90 mmol) and phosphorousoxychloride (1.89 ml, 20 mmol) in 50 ml of anhydrous acetonitrile. Tothe resulting suspension the solution of 2′,3′,5′-tri-O-Benzoyl-6-methyluridine (5.7 g, 10 mmol) in 30 ml of acetonitrile was added dropwise andthe reaction mixture was stirred for 4 hours at room temperature. Thenit was concentrated in vacuo to minimal volume (not to dryness). Theresidue was dissolved in chloroform and washed with water, saturated aqsodium bicarbonate and brine. The organic layer was dried over sodiumsulfate and the solvent was removed in vacuo. The residue was dissolvedin 100 ml of 1,4-dioxane and treated with 50 mL of 29% aq NH₄OHovernight. The solvents were removed in vacuo. The residue was dissolvedin the in the mixture of pyridine (60 mL) and methanol (10 mL), cooledto −15° C. and ice-cooled 2M aq solution of sodium hydroxide was addedunder stirring. The reaction mixture was stirred at -10 to -15° C. foradditional 30 minutes and then neutralized to pH 7 with Dowex 50 (Py⁺).The resin was filtered off and washed with 200 mL of the mixture H₂O—Py(4:1). The combined mother liquor and washings were evaporated todryness. The residue was cristallized from aq methanol to give 1.6 g(62%) of 6-methyl cytidine.

To the solution of 6-methyl cytidine (1.4 g, 5.44 mmol) in dry pyridine3.11 mL of trimethylchlorosilane was added and the reaction mixture wasstirred for 2 hours at room temperature. Then acetic anhydride (0.51 mL,5.44 mmol) was added and the reaction mixture was stirred for additional3 hours at room temperature. TLC showed disappearance of the startingmaterial and the reaction was quenched with MeOH (20 mL), ice-cooled andtreated with water (20 mL, 1 hour). The solvents wee removed in vacuoand the residue was dried by four coevaporations with dry pyridine.Finally it was redissolved in dry pyridine and dimethoxytrityl chloride(2.2 g, 6.52 mmol) was added. The reaction mixture was stirred overnightat room temperature and quenched with MeOH (20 mL). The solvents wereremoved in vacuo. The remaining oil was dissolved in methylene chloride,washed with saturated sodium bicarbonate and brine. The organic layerwas separated and evaporated and the residue was purified by flashchromatography on silica gel with the gradient of MeOH in methylenechloride (3% to 5%) to give 2.4 g (74%) of the compound (4).

Example 9

Synthesis of 6-aza-uridine and 6-aza-cytidine

To the solution of 6-aza uridine (5 g, 20.39 mmol) in dry pyridinedimethoxytrityl chloride (8.29 g, 24.47 mmol) was added and the reactionmixture was left overnight at room temperature. Then it was quenchedwith methanol (50 mL) and the solvents were removed in vacuo. Theremaining oil was dissolved in methylene chloride and washed withsaturated aq sodium bicarbonate and brine. The organic layer wasseparated and evaporated to dryness. The residue was additionally driedby multiple coevaporations with dry pyridine and finally dissolved indry pyridine. Acetic anhydride (4.43 mL, 46.7 mmol) was added to theabove solution and the reaction mixture was left for 3 hours at roomtemperature. Then it was quenched with methanol and worked-up as above.The residue was purified by flash chromatography on silics gel usingmixture of 2% of MeOH in methylene chloride as an eluent to give 9.6 g(75%) of the compound.

Triethylamine (23.7 ml, 170.4 mmol) was added dropwise to a stirredice-cooled mixture of 1,2,4-triazole (10.6 g, 153.36 mmol) andphosphorous oxychloride (3.22 ml, 34.08 mmol) in 100 ml of anhydrousacetonitrile. To the resulting suspension the solution of2′,3′-di-O-Acetyl-5′-O-Dimethoxytrityl-6-aza Uridine (7.13 g, 11.36mmol) in 40 ml of acetonitrile was added dropwise and the reactionmixture was stirred for 6 hours at room temperature. Then it wasconcentrated in vacuo to minimal volume (not to dryness). The residuewas dissolved in chloroform and washed with water, saturated aq sodiumbicarbonate and brine. The organic layer was dried over sodium sulfateand the solvent was removed in vacuo. The residue was dissolved in 150ml of 1,4-dioxane and treated with 50 mL of 29% aq NH₄OH for 20 hours atroom temperature. The solvents were removed in vacuo. The residue waspurified by flash chromatigraphy on silica gel using linear gradient ofMeOH (4% to 10%) in methylene chloride as an eluent to give 3.lg (50%)of azacytidine.

To the stirred solution of 5′-O-Dimethoxytrityl-6-aza cytidine (3 g,5.53 mmol) in anhydrous pyridine trimethylchloro silane (2.41 mL, 19mmol) was added and the reaction mixture was left for 4 hours at roomtemperature. Then acetic anhydride (0.63 mL, 6.64 mmol) was added andthe reaction mixture was stirred for additional 3 hours at roomtemperature. After that it was quenched with MeOH (15 mL) and thesolvents were removed in vacuo. The residue was treated with 1M solutionof tetrabutylammonium fluoride in THF (20°, 30 min) and evaporated todryness. The remaining oil was dissolved in methylene chloride, washedwith saturated aq sodium bicarbonate and water. The separated organiclayer aws dried over sodium sulfate and evaporated to dryness. Theresidue was purified by flash chromatography on silica gel using 4% MeOHin methylene chloride as an eluent to give 2.9 g (89.8%) of thecompound.

General Procedure for the Introducing of the TBDMS-Group: To the stirredsolution of the protected nucleoside in 50 mL of dry THF and pyridine (4eq) AgNO₃ (2.4 eq) was added. After 10 minutes tert-butyidimethylsilylchloride (1.5 eq) was added and the reaction mixture was stirred at roomtemperature for 12 hours. The resulted suspension was filtered into 100mL of 5% aq NaHCO₃. The solution was extracted with dichloromethane(2×100 mL). The combined organic layer was washed with brine, dried overNa₂SO₄ and evaporated. The residue was purified by flash chromatographyon silica gel with hexanes-ethylacetate (3:2) mixture as eluent.

General Procedure for Phosphitylation: To the ice-cooled stirredsolution of protected nucleoside (1 mmol) in dry dichloromethane (20 mL)under argon blanket was added dropwise via syringe the premixed solutionof N,N-diisopropylethylamine (2.5eq) and 2-cyanoethylN′N-diisopropylchlorophosphoramidite (1.2 eq) in dichloromethane (3 mL).Simultaneously via another syringe N-methylimidazole (1 eq) was addedand stirring was continued for 2 hours at room tenperature. After thatthe reaction mixture was again ice-cooled and quenched with 15 ml of drymethanol. After 5 min stirring, the mixture was concentrated in vacuo(<40° C.) and purified by flash chromatography on silica gel usinghexanes-ethylacetate mixture contained 1% triethylamine as an eluent togive corresponding phosphoroamidite as white foam.

Example 10

RNA cleavage activity of HHA ribozyme substituted with 6-methyl-Uridine

Hammerhead ribozymes targeted to site A (see FIG. 15) were synthesizedusing solid-phase synthesis, as described above. U4 position wasmodified with 6-methyl-uridine.

RNA cleavage assay in vitro:

Substrate RNA is 5′ end-labeled using [γ-³²P] ATP and T4 polynucleotidekinase (US Biochemicals). Cleavage reactions were carried out underribozyme “excess” conditions. Trace amount (≦1 nM) of 5′ end-labeledsubstrate and 40 nM unlabeled ribozyme are denatured and renaturedseparately by heating to 90° C. for 2 min and snap-cooling on ice for10-15 min. The ribozyme and substrate are incubated, separately, at 37°C. for 10 min in a buffer containing 50 mM Tris-HCl and 10 mM MgCl₂. Thereaction is initiated by mixing the ribozyme and substrate solutions andincubating at 37° C. Aliquots of 5 μl are taken at regular intervals oftime and the reaction is quenched by mixing with equal volume of 2Xformamide stop mix. The samples are resolved on 20 % denaturingpolyacrylamide gels. The results are quantified and percentage of targetRNA cleaved is plotted as a function of time.

Referring to FIG. 16, hammerhead ribozymes containing 6-methyl-uridinemodification at U4 position cleave the target RNA efficiently.

Example 11

RNA cleavage activity of HHB ribozyme substituted with 6-methyl-Uridine

Hammerhead ribozymes targeted to site B (see FIG. 17) were synthesizedusing solid-phase synthesis, as described above. U4 and U7 positionswere modified with 6-methyl-uridine.

RNA cleavage reactions were carried out as described above. Referring toFIG. 18, hammerhead ribozymes containing 6-methyl-uridine modificationat U4 and U7 positions cleave the target RNA efficiently.

Example 12

RNA cleavage activity of HHC ribozyme substituted with 6-methyl-Uridine

Hammerhead ribozymes targeted to site C (see FIG. 19) were synthesizedusing solid-phase synthesis, as described above. U4 and U7 positionswere modified with 6-methyl-uridine.

RNA cleavage reactions were carried out as described above. Referring toFIG. 20, hammerhead ribozymes containing 6-methyl-uridine modificationat U4 positions cleave the target RNA efficiently.

Sequences listed in FIGS. 7, 15, 17 19 and the modifications describedin these figures are meant to be non-limiting examples. Those skilled inthe art will recognize that variants (base-substitutions, deletions,insertions, mutations, chemical modifications) of the ribozyme and RNAcontaining other 2′-hydroxyl group modifications, including but notlimited to amino acids, peptides and cholesterol, can be readilygenerated using techniques known in the art, and are within the scope ofthe present invention.

Example 13

Inhibition of Rat smooth muscle cell proliferation by 6-methyl-Usubstituted ribozyme HHA.

Hammerhead ribozyme (HHA) is targeted to a unique site (site A) withinc-myb mRNA. Expression of c-myb protein has been shown to be essentialfor the proliferation of rat smooth muscle cell (Brown et al., 1992 J.Biol. Chem. 267, 4625).

The ribozymes that cleaved site A within c-myb RNA described above wereassayed for their effect on smooth muscle cell proliferation. Ratvascular smooth muscle cells were isolated and cultured as described(Stinchcomb et al., supra). HHA ribozymes were complexed with lipids anddelivered into rat smooth muscle cells. Serum-starved cells werestimulated as described by Stinchcomb et al., supra. Briefly,serum-starved smooth muscle cells were washed twice with PBS, and theRNA/lipid complex was added. The plates were incubated for 4 hours at37° C. The medium was then removed and DMEM containing 10% FBS,additives and 10 μM bromodeoxyuridine (BrdU) was added. In some wells,FBS was omitted to determine the baseline of unstimulated proliferation.The plates were incubated at 37° C. for 20-24 hours, fixed with 0.3%H₂O₂ in 100% methanol, and stained for BrdU incorporation by standardmethods. In this procedure, cells that have proliferated andincorporated BrdU stain brown; non-proliferating cells arecounter-stained a light purple. Both BrdU positive and BrdU negativecells were counted under the microscope. 300-600 total cells per wellwere counted. In the following experiments, the percentage of the totalcells that have incorporated BrdU (% cell proliferation) is presented.Errors represent the range of duplicate wells. Percent inhibition thenis calculated from the % cell proliferation values as follows: %inhibition=100×100 (Ribozyme−0% serum)/(Control−0% serum).

Referring to FIG. 21, active ribozymes substituted with 6-methyl-U atposition 4 of HHA were successful in inhibiting rat smooth muscle cellproliferation. A catalytically inactive ribozyme (inactive HHA), whichhas two base substitutions within the core (these mutations inactivate ahammerhead ribozyme; Stinchcomb et al., supra), does not significantlyinhibit rat smooth muscle cell proliferation.

Example 14

Inhibition of stromelysin production in human synovial fibroblast cellsby 6-methyl-U substituted ribozyme HHC.

Hammerhead ribozyme (HHC) is targeted to a unique site (site C) withinstromelysin mRNA.

The general assay was as described (Draper et al., supra). Briefly,fibroblasts, which produce stromelysin, are serum-starved overnight andribozymes or controls are offered to the cells the next day. Cells weremaintained in serum-free media. The ribozyme were applied to the cellsas free ribozyme, or in association with various delivery vehicles suchas cationic lipids (including Transfectam™, Lipofectin™ andLipofectamine™), conventional liposomes, non-phospholipid liposomes orbiodegradable polymers. At the time of ribozyme addition, or up to 3hours later, lnterleukin-1α (typically 20 units/ml) can be added to thecells to induce a large increase in stromelysin expression. Theproduction of stromelysin can then be monitored over a time course,usually up to 24 hours.

Supernatants were harvested 16 hours after IL-1 induction and assayedfor stromelysin expression by ELISA. Polyclonal antibody against MatrixMetalloproteinase 3 (Biogenesis, NH) was used as the detecting antibodyand anti-stromelysin monoclonal antibody was used as the capturingantibody in the sandwich ELISA (Maniatis et al., supra) to measurestromelysin expression.

Referring to FIG. 22, HHC ribozyme containing 6-methyl-U modification,caused a significant reduction in the level of stromelysin proteinproduction. Catalytically inactive HHC had no significant effect on theprotein level.

Other embodiments are within the following claims.

TABLE I Characteristics of Ribozymes Group I Introns Size: ˜200 to >1000nucleotides. Requires a U in the target sequence immediately 5′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of cleavage site. Over75 known members of this class. Found in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portionof a ribonucleoprotein enzyme. Cleaves tRNA precursors to form maturetRNA. Roughly 10 known members of this group all are bacterial inorigin. Hammerhead Ribozyme Size: ˜13 to 40 nucleotides. Requires thetarget sequence UH immediately 5′ of the cleavage site. Binds a variablenumber nucleotides on both sides of the cleavage site. 14 known membersof this class. Found in a number of plant pathogens (virusoids) that useRNA as the infectious agent (FIG. 1) Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of the cleavage site anda variable number to the 3′ side of the cleavage site. Only 3 knownmember of this class. Found in three plant pathogen (satellite RNAs ofthe tobacco ringspot virus, arabis mosaic virus and chicory yellowmottle virus) which uses RNA as the infectious agent (FIG. 3). HepatitisDelta Virus (HDV) Ribozyme Size: 50-60 nucleotides (at present).Cleavage of target RNAs recently demonstrated. Sequence requirements notfully determined. Binding sites and structural requirements not fullydetermined, although no sequences 5′ of cleavage site are required. Only1 known member of this class. Found in human HDV (FIG. 4). Neurospora VSRNA Ribozyme Size: ˜144 nucleotides (at present) Cleavage of target RNAsrecently demonstrated. Sequence requirements not fully determined.Binding sites and structural requirements not fully determined. Only 1known member of this class. Found in Neurospora VS RNA (FIG. 5).

12 1 11 RNA Artificial Sequence Description of Artificial SequenceSynthesized Hammerhead Target. 1 nnnnuhnnnn n 11 2 28 RNA ArtificialSequence Description of Artificial Sequence Synthesized HammerheadRibozyme. 2 nnnnncugan gagnnnnnnc gaaannnn 28 3 15 RNA ArtificialSequence Description of Artificial Sequence Synthesized Hairpin Target.3 nnnnnnnyng hynnn 15 4 47 RNA Artificial Sequence Description ofArtificial Sequence Synthesized Hairpin Ribozyme. 4 nnnngaagnnnnnnnnnnna aahannnnnn nacauuacnn nnnnnnn 47 5 85 RNA Artificial SequenceDescription of Artificial Sequence Hepatitis Delta Virus (HDV) Ribozyme.5 uggccggcau ggucccagcc uccucgcugg cgccggcugg gcaacauucc gaggggaccg 60uccccucggu aauggcgaau gggac 85 6 176 RNA Artificial Sequence Descriptionof Artificial Sequence Neurospora VS RNA Enzyme. 6 gggaaagcuu gcgaagggcgucgucgcccc gagcgguagu aagcagggaa cucaccucca 60 auuucaguac ugaaauugucguagcaguug acuacuguua ugugauuggu agaggcuaag 120 ugacgguauu ggcguaagucaguauugcag cacagcacaa gcccgcuugc gagaau 176 7 15 RNA Artificial SequenceDescription of Artificial Sequence Target of Hammerhead Ribozyme to SiteA (HHA). 7 ggagaauugg aaaac 15 8 34 RNA Artificial Sequence Descriptionof Artificial Sequence Hammerhead Ribozyme to Site A (HHA). 8 guuuucccngaugaggcgaa agccgaaauu cucc 34 9 15 RNA Artificial Sequence Descriptionof Artificial Sequence Target of Hammerhead Ribozyme to Site B (HHB). 9agggauuaau ggaga 15 10 34 RNA Artificial Sequence Description ofArtificial Sequence Target of Hammerhead Ribozyme to Site B (HHB). 10ucuccaucng angaggcgaa agccgaaaau cccu 34 11 15 RNA Artificial SequenceDescription of Artificial Sequence Target of Hammerhead Ribozyme to SiteC (HHC). 11 cuguuuuuga agaau 15 12 34 RNA Artificial SequenceDescription of Artificial Sequence Target of Hammerhead Ribozyme to SiteC (HHC). 12 auucuuccng angaggcgaa agccgaaaau acag 34

What is claimed is:
 1. An enzymatic nucleic acid molecule with RNAcleaving activity comprising one or more ribonucleotides, nucleotidebase modifications, and sugar modifications, wherein said nucleotidebase modification is 6-dimethylaminopurine, 6-methylaminopurine,6-amino-8-bromopurine, 6-amino-8-fluoropurine, 6-methyluracil, 3-methyluracil, 5,6-dihydrouracil, 6-azauracil, 8-bromoguanine,8-fluoroguanine, N4,N4-dimethcytosine, N4-methylcytosine, or 2-pyridone.2. The enzymatic nucleic acid molecule of claim 1, wherein saidribonucleotides are present in the cataytic core of the enzymaticnucleic acid molecule.
 3. The enzymatic acid molecule of claim 1,wherein said nucleotide base modification is present in the catalyticcore of the enzymatic nucleic acid molecule.
 4. The enzymatic nucleicacid molecule of claim 1, wherein said nucleotide base modification ispresent in a binding arm of the enzymatic nucleic acid molecule.
 5. Theenzymatic nucleic acid molecule of claim 1, wherein said sugarmodification is present in the catalytic core of the enzymatic nucleicacid molecule.
 6. The enzymatic nucleic acid molecule of claim 1,wherein said sugar modification is present in a binding arm of theenzymatic nucleic acid molecule.
 7. The enzymatic nucleic acid moleculeof claim 1, wherein said ribonucleotides, the nucleotide basemodification and the sugar modification are present in the catalyticcore of the enzymatic nucleic acid molecule.
 8. The enzymatic nucleicacid molecule of claim 1, wherein said enzymatic nucleic acid moleculeis in a hammerhead motif.